Methamphetamine Vaccines: Improvement through Hapten Design

Article abstract of DOI:10.1021/acs.jmedchem.6b00084

Methamphetamine (MA) addiction is a serious public health problem, and current methods to abate addiction and relapse are currently ineffective for mitigating this growing global epidemic. Development of a vaccine targeting MA would provide a complementary strategy to existing behavioral therapies, but this has proven challenging. Herein, we describe optimization of both hapten design and formulation, identifying a vaccine that elicited a robust anti-MA immune response in mice, decreasing methamphetamine-induced locomotor activity.

Full text of DOI:10.1021/acs.jmedchem.6b00084

Article
pubs.acs.org/jmc
Methamphetamine Vaccines: Improvement through Hapten Design
†
†,‡
†
,†
Karen C. Collins, Joel E. Schlosburg, Paul T. Bremer, and Kim D. Janda*
†
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine
WIRM) and Committee on Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 North Torrey Pines Road,
‡
(
La Jolla, California 92037, United States
*
S Supporting Information
ABSTRACT: Methamphetamine (MA) addiction is a serious
public health problem, and current methods to abate addiction
and relapse are currently ineffective for mitigating this growing
global epidemic. Development of a vaccine targeting MA
would provide a complementary strategy to existing behavioral
therapies, but this has proven challenging. Herein, we describe
optimization of both hapten design and formulation,
identifying a vaccine that elicited a robust anti-MA immune
response in mice, decreasing methamphetamine-induced locomotor activity.
INTRODUCTION
rigor of direct comparison with successful haptens from both
our laboratory and others.
■
Methamphetamine (MA) is a highly addictive stimulant;
administration of the drug results in an intense, euphoric
high created by the release of multiple neurotransmitters in the
brain. Frequent MA use leads to dependence, and attempted
abstinence in dependent users precipitates severe withdrawal
We have previously designed hapten 1, linked to the carrier
protein via the N-methyl group, and shown that this can
stimulate moderate affinities and concentrations of anti-MA
antibodies, with an observed decrease in MA-induced
23
locomotor activity (Figure 1). In this case we had selected
1
−3
and a correspondingly high relapse rate.
There are no
a thiol-maleimide linkage due to the presence of a secondary
amine in the structure, in order to ensure selectivity in the
hapten−protein conjugation. However, we have observed that
thiol addition to maleimide-activated proteins typically results
in lower hapten densities than amide bond formation, with
haptens that are less stable than their acid counterparts.
Previous data had also demonstrated that the tertiary amine
hapten 3 was moderately effective at eliciting anti-MA
4
,5
approved medications to treat MA addiction, with behavioral
therapies providing only limited improvement to long-term
6
,7
abstinence rates. With MA use (and the corresponding social
8
,9
and economic burden) on the rise globally, it is vital that new
treatments are discovered that can increase the chances of long-
term abstinence. A vaccine against MA would decrease the
likelihood of relapse through the sequestration of the stimulant
from the bloodstream, limiting its psychoactive effects.
However, MA, as other small molecules, cannot be recognized
by the immune system alone. Conjugation to an immunogenic
carrier is required to enable effective processing and recognition
by leukocytes and ultimately the production of a long-lasting
26
antibodies, and we wanted to clarify whether modification
of the amine had an impact on the resulting immune response
through direct comparison with its secondary amine hapten
counterpart, 2. Additionally, we have recently found that the
introduction of a diglycine linker into a nicotine hapten
enhances both concentration and affinity of the resulting
1
0
antibody response. This strategy has shown promise for both
27
11−17
antibodies, with a postulated enhancement of antibody titers
against oxycodone and hydrocodone upon the inclusion of
nicotine and cocaine, with vaccines reaching clinical trials,
but an application to treat MA abuse is still in its infancy.
18
28,29
diglycine and tetraglycine linkers.
Thus, we targeted hapten
To sequester MA successfully, a vaccine must elicit a high
concentration of high-affinity MA-specific antibodies. The
affinity and specificity of the antibodies generated is strongly
dependent on identifying an effective hapten, which is a
significant challenge given the small size of the drug and its
limited chemical epitope. A range of MA haptens have been
4
as our first glycine-containing hapten. We also endeavored to
directly compare our haptens with 5, a hapten that has been
reported by Kosten and Orson to reduce MA-induced hyper-
and hypolocomotion in mice that is currently progressing to
25,30
clinical trials.
19−26
reported by various groups,
but the drugs of abuse vaccine
RESULTS AND DISCUSSION
field suffers from variability in a number of important
methodological factors that makes intrastudy hapten design
comparison innately flawed. These factors include carrier
protein, adjuvant, formulation, vaccination schedule, and
analytical methods, among others. We therefore sought to
further our optimization of MA haptens but with the added
■
We began by synthesizing MA haptens 2 and 3 by alkylation
26
of amphetamine and MA, respectively, followed by ester
Received: January 19, 2016
©
XXXX American Chemical Society
A
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Figure 1. Structures of haptens 1−5.
Scheme 1. Synthesis of Haptens 2−5
Figure 2. Anti-MA antibody titers, affinities, and concentrations from 2-DT, 3-DT, 4-DT, and 5-DT vaccinated mice (n = 6) with SAS as adjuvant.
a) Midpoint titers as determined by ELISA using 1-BSA as the coating antigen; (b) dissociation constants (K ’s) as determined by competitive RIA
(
d
using pooled sera; and (c) antibody concentrations as determined by competitive RIA using pooled sera (63 days). RIA data were obtained in
duplicate. Errors represent SEM.
B
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Scheme 2. Synthesis of Haptens 9−12
Figure 3. Anti-MA antibody titers, affinities, and concentrations from 2-DT (with SAS or alum), 9-DT, 10-DT, and 12-DT vaccinated mice (n = 6)
with SAS (or alum) as adjuvant. (a) Midpoint titers as determined by ELISA using 1-BSA as the coating antigen; (b) dissociation constants (K ’s) as
d
determined by competitive RIA using pooled sera; and (c) antibody concentrations as determined by competitive RIA using pooled sera (63 days).
RIA data were obtained in triplicate. Errors represent SEM.
hydrolysis (Scheme 1). The diglycine variant, 4, could be
antigen (Figure 2a). Titers induced by 2-DT, 3-DT, and 4-DT
were similar over the course of the vaccination schedule. The
titers for 5-DT-vaccinated mice were significantly lower,
although this could be partly attributed to the difference
between the structures of the coating antigen and the hapten.
Radioimmunoassay (RIA) analysis was performed to
determine the antibody concentrations and their affinity for
MA itself, enabling unbiased comparison of the four haptens
(Figure 2b,c). 2-DT and 4-DT elicited antibodies with
comparable affinity for MA, but the presence of the peptidic
linker gave a moderate boost to the antibody concentration,
paralleling results previously observed for the aforementioned
synthesized using an analogous method to the strategy for
27
nicotine hapten AM1(Gly)2. Alkylation of amphetamine with
31
5
-((tert-butoxycarbonyl)amino)pentyl methanesulfonate and
Boc-deprotection, followed by coupling with Boc-Gly-Gly-OH
gave carbamate 7, which was subsequently deprotected and
coupled with monomethyl glutarate before ester hydrolysis.
Hapten 5 was prepared as previously reported, by reaction of
25
MA with succinic anhydride.
The four haptens were activated and conjugated to
diphtheria toxoid (DT), an immunogenic protein commonly
used as a carrier for conjugate vaccine haptens, with comparable
conjugation efficiencies as determined by MALDI-TOF (Table
S1). LCMS analysis confirmed that neither 2 nor 4 polymerized
under extended activation times. Furthermore, the comparable
hapten densities resulting from conjugation of 2 and 3 suggests
that lactamization was not a significant side-reaction.
27
peptidic nicotine hapten. Antibodies raised against 3-DT had
much poorer MA affinity, demonstrating the detrimental effect
of modification of the secondary amine to a tertiary amine. This
hypothesis is further supported by the dramatic reduction in
MA-binding capabilities observed for antibodies raised against
5-DT, the design of which converted the crucial secondary
amine, protonated under physiological pH, to an uncharged
tertiary amide. Full analysis for this hapten was not possible
because of the low MA binding obtained; the highest serum
These four conjugates were formulated with Sigma Adjuvant
System (SAS), an oil-in-water emulsion adjuvant containing a
Toll-like receptor 4 agonist, monophosphoryl lipid A (MPLA).
SAS has been effectively used in previous campaigns to evaluate
27,32−34
3
drugs of abuse vaccines.
Once formulated, the efficacies
concentration tested bound only 7.4% H-MA tracer, compared
of the four vaccines were determined by vaccination of Swiss
to 48.5% for 2-DT-elicited antibodies (Graph S1).
Webster mice (n = 6). Antibody titers of mouse serum samples
were determined by ELISA using 1-BSA as the coating
The ultimate goal of the MA vaccine is the ability to protect
against MA administration in vivo. However, no modulation of
22
C
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MA-induced hyperlocomotion in mice was observed for any of
these four vaccines. The presence of a secondary amine in the
hapten appeared crucial for obtaining an effective immune
response, and we thus focused in the next round of
optimization on modulating the linker.
The introduction of a peptidic linker resulted in a moderate
increase in antibody concentration, yet our first design concept
required a lengthy synthesis, with an additional glutarate moiety
necessary for conjugation. We therefore investigated simpler
glycine and diglycine variants, with amides in the reverse
direction to allow for direct conjugation to the carrier protein.
Additionally, given that natural, peptidic linkers appear to
generate a greater immune response, we targeted a hapten with
a shorter alkyl spacer between the MA core and the glycine
unit.
CpG ODN 1826 and again used to vaccinate Swiss Webster
mice (n = 6). On this occasion, the corresponding vaccine with
unconjugated TT was used as a control for behavioral analysis.
The resulting ELISA analysis showed exceptionally high
titers, suggesting that this new vaccine formulation was
extremely effective at eliciting an anti-MA immune response
(Figure 4a). The new formulation succeeded in stimulating
Haptens 9 and 10 were synthesized from 2 by coupling with
the protected esters of glycine and diglycine, respectively,
followed by saponification to release the acids (Scheme 2).
Hapten 11 was synthesized directly from amphetamine through
alkylation with tert-butyl 3-bromopropanoate and acid-medi-
t
ated ester cleavage. Coupling with H-Gly-O Bu and subsequent
deprotection then generated hapten 12.
The four haptens 9−12 were activated, along with hapten 2
as a control, and conjugated to diphtheria toxoid (DT). Hapten
Figure 4. Anti-MA antibody titers, affinities and concentrations from
1
1 failed to conjugate because of immediate cyclization to the
1
2-TT vaccinated mice (n = 6) with alum/CpG ODN 1826 as
corresponding azetidinone. The three reverse-glycine-contain-
ing haptens conjugated to the carrier protein with efficiency
comparable to that of hapten 2, suggesting that lactamization
was not occurring.
adjuvant. (a) Midpoint titers as determined by ELISA using 1-BSA as
the coating antigen; (b) dissociation constants (K ’s) as determined by
d
competitive RIA using pooled sera; and (c) antibody concentrations as
determined by competitive RIA using pooled sera (63 days). RIA data
were obtained in triplicate. Errors represent SEM.
The four successful conjugates were formulated with SAS,
and 2-DT was also separately formulated with alum for
comparison of adjuvants. Alum is a clinically approved adjuvant
found in most human vaccines and is effective for boosting
humoral responses to target antigens. The efficacies of the
vaccines were again determined by vaccination of Swiss
Webster mice (n = 6). All four SAS formulations resulted in
similar titers, with a slight increase using alum (Figure 3a). RIA
analysis again allowed for unbiased hapten comparison, with
similar antibody affinities and concentrations for the SAS
formulations of 2-DT, 9-DT, and 10-DT (Figure 3b,c).
Excitingly, 12-DT elicited significantly higher affinity antibod-
ies, suggesting that we had identified a superior hapten design.
Unfortunately, a significantly lower antibody concentration was
obtained, but this variable could be more readily improved
through adjuvant formulation optimization. In this study, we
found that formulating 2-DT with alum resulted in higher
antibody concentrations compared to SAS with a slight
reduction in antibody affinity, and therefore a combination of
higher antibody concentrations, as determined by RIA, albeit
with an unexpected reduction in antibody affinity (Figure 4b,c).
However, the true test of an MA vaccine is its ability to reduce
an in vivo response to MA administration.
The effects of our lead MA vaccine were examined in a
standard behavioral model for MA psychoactivity. Because MA
is a stimulant, it produces a quantifiable increase in rodent
locomotor activity. By using a video tracking system, we
measured MA-induced locomotor activity in both vaccinated
and control mice (n = 6). In the control group, a clear dose−
response curve was observed at a wide range of MA doses
(0.5−4.0 mg/kg); as expected, MA dosing varied directly with
locomotor activity and inversely with time immobile (Figure
5a,b). Gratifyingly, 12-TT showed a significant shift in the
dose−response curve for both locomotor activity [dose ×
vaccine interaction: F3,30 = 4.88, p < 0.01] and immobility time
[dose × vaccine interaction: F4,40 = 2.85, p < 0.05], especially at
2 mg/kg, thus demonstrating a marked vaccine-mediated
attenuation of the effects of MA. We attribute this successful
result to a combination of successful MA hapten design and
adjuvant formulation.
1
2-DT with alum was our starting point for further
optimization.
A decrease in ELISA titer was observed between days 28 and
4
2 for both studies, suggesting that improvements could be
made to the vaccine formulation and schedule. In previous
work, the use of CpG ODN 1826 has proved highly effective at
increasing the immune response obtained for our heroin
CONCLUSIONS
■
35
vaccine over using alum alone, and the use of tetanus toxoid
TT) rather than DT was preferred because of the potential for
A series of comparative studies have demonstrated the
importance of hapten design for drugs of abuse vaccines; an
immune response will only be directed toward the drug of
interest should the hapten be a true representation of the drug.
In the case of MA, preservation of the secondary amine was
essential for eliciting high-affinity anti-MA antibodies; haptens
containing a tertiary amine (hapten 3) or an amide (hapten
(
higher hapten densities. All of these components were therefore
brought together for our final vaccination study. Hapten 12 was
activated and conjugated to TT, resulting in a high hapten
density far greater than that attainable for DT because of the
increased number of lysine residues present on the surface of
the larger protein. The vaccine was formulated with alum and
2
5,30
5)
failed to produce effective drug-binding antibodies.
D
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Journal of Medicinal Chemistry
Article
Figure 5. MA-induced locomotor activity in 12-TT vaccinated and TT-only control mice (n = 6) using alum/CpG ODN 1826 as adjuvant. (a)
Change in overall locomotor activity due to various doses of MA. Activity was measured as raw distances traveled using video tracking software for
9
0 min following injection and represented as percent change from distance traveled during saline control session. (b) Time spent immobile during
locomotor sessions. Errors represent SEM *, p < 0.05 and ***, p < 0.001 versus TT control group, analyzed using Prism 6 software, using a two-way
repeated-measures ANOVA with Bonferroni posthoc-corrected comparisons.
Additionally, the superiority of peptidic over alkyl linkers to
generate effective conjugates was further supported. The choice
of carrier protein and vaccine formulation were key in obtaining
an efficacious vaccine in vivo, with 12-TT/alum+CpG
attenuating MA-induced locomotion in mice. This vaccine is
therefore a highly promising candidate for further development,
with the ultimate aim of human clinical trials.
118.16 (q, J = 292.5 Hz), 56.9, 46.1, 40.3, 34.4, 27.2, 27.0, 25.4, 16.0.
Found: 250.1803. C H NO requires 250.1801.
15 23
2
(S)-N1-(1-Phenylpropan-2-yl)pentane-1,5-diamine, 6. A sol-
ution of D-amphetamine·0.5H SO (200 mg, 1.09 mmol), 5-((tert-
2
4
3
1
butoxycarbonyl)amino)pentyl methanesulfonate (458 mg, 1.63
mmol), and K CO (450 mg, 3.26 mmol) in MeCN (6 mL) was
2
3
heated to 80 °C (24 h), whereupon the solution was cooled to rt and
diluted with CH Cl (20 mL). The solution was washed with brine (20
2
2
mL), the aqueous layer extracted with CH Cl (2 × 20 mL), the
2
2
EXPERIMENTAL SECTION
combined organic layers dried (MgSO ) and filtered, and the solvent
4
■
removed in vacuo. Purification by preparative RP-HPLC (C18
Reactions were carried out under an argon atmosphere at rt using
flame-dried glassware with dry solvents. Reagents were used as
commercially supplied, unless otherwise specified. RP-HPLC was
performed on an Agilent Technologies 1260 Infinity system using a
Grace Vydac C18 column, 10−15 μm, 250 × 22 mm using the
column, R = 39.9 min) followed by lyophilization gave intermediate
t
25
carbamate as a colorless oil (192 mg, 41%), [α]D + 9.2 (c 2.0, CHCl );
3
δ (CDCl , 600 MHz) 9.22 (s, 2H), 7.30 (t, J = 7.4 Hz, 2H), 7.24 (t, J
H
3
=
7.3 Hz, 1H), 7.15 (d, J = 7.2 Hz, 2H), 3.39−3.32 (m, 1H), 3.29 (dd,
J = 13.1, 3.6 Hz, 1H), 3.06−2.97 (m, 3H), 2.97−2.90 (m, 1H), 2.68
following method: [A = 0.1% TFA/H O, B = 0.1% TFA/MeCN; λ =
2
(
1
1
dd, J = 12.8, 10.8 Hz, 1H), 1.78−1.71 (m, 2H), 1.46−1.39 (m, 11H),
2
54 and 210 nm; gradient 1% B (5 min), 1−15% B (15 min), 15−75%
.39−1.33 (m, 2H), 1.23 (d, J = 6.5 Hz, 3H); δ (CDCl , 151 MHz)
B (35 min), 75−95% B (10 min), 95% B (5 min)]. Flash
C
3
61.8 (q, J = 37.4, 36.7 Hz), 156.3, 136.1, 127.4, 116.4 (q, J = 292.1
chromatography was performed on silica gel (SiliaFlash P60 40−63
3
6
Hz), 79.4, 56.1, 45.1, 40.1, 39.6, 29.6, 28.5, 25.9, 23.8, 15.5. Found:
21.2536. C H N O requires 321.2536. A solution of intermediate
μm, 230−400 mesh) according to the method of Still et al. TLC was
3
performed on glass-backed plates precoated with silica (EMD 60 F254,
19 33
2
2
carbamate (50 mg, 0.115 mmol) in 4 M HCl in dioxane (1.5 mL) was
0
.25−1 mm) and developed using standard visualizing agents: UV
stirred at rt (1.5 h) before the solvent was removed in vacuo to give
fluorescence (254 nm) and KMnO , cerium ammonium nitrate, or
4
2
5
1
13
the title compound as a pale yellow oil (34 mg, 100%), [α] + 5.8 (c
ninhydrin with appropriate heating. H and C NMR were performed
D
2
.0, MeOH); δ (CD OD, 600 MHz) 7.34 (t, J = 7.0 Hz, 2H), 7.29
on Bruker and Varian spectrometers, with the reference from the
H 3
1
(d, J = 7.3 Hz, 2H), 7.27 (t, J = 7.0 Hz, 1H), 3.74 (t, J = 5.5 Hz, 1H),
residual solvent peak for H NMR (7.26 ppm for CDCl , 3.31 ppm for
3
13
3
1
1
.69−3.65 (m, 1H), 3.58 (t, J = 4.6 Hz, 1H), 3.52 (s, 1H), 3.30 (s,
CD OD) and the solvent peak for C NMR (77.1 ppm for CDCl3,
3
H), 3.11 (s, 2H), 2.98 (s, 1H), 2.75 (t, J = 10.6 Hz, 2H), 1.82 (s, 2H),
4
9.0 ppm for CD OD). HRMS (ESI, positive ion mode) was
3
.76 (s, 2H), 1.54 (s, 2H), 1.24 (s, 3H); δC (CD OD, 151 MHz,
performed on an Agilent 1100 Deries LC/MSD-TOF, protein
concentration was determined by BCA assay (Pierce BCA Protein
Assay Kit) with analysis on a plate reader (Molecular Devices
3
mixture of rotamers) 137.4, 130.5, 129.9, 128.2, 73.5, 72.4, 62.1, 57.1,
46.1, 43.8, 40.5, 40.2, 27.9, 26.9, 24.5, 16.0. Found: 221.2013.
37
SpectraMax 250) at 562 nm, and determination of copy number of
protein conjugates was carried out using comparative MALDI-MS,
using sinapinic acid as the matrix on an Applied Biosystems Voyager-
C H N requires 221.2012.
14 25 2
tert-Butyl (S)-(2-Oxo-2-((2-oxo-2-((5-((1-phenylpropan-2-yl)-
amino)pentyl)amino)ethyl)amino)ethyl)carbamate, 7. A solu-
tion of amine 6 (16 mg, 0.0546 mmol), Boc-Gly-Gly-OH (14 mg,
38
DE STR MS. All compounds had purity >95%.
S)-6-((1-Phenylpropan-2-yl)amino)hexanoic Acid, 2. Ethyl 6-
0
0
.0600 mmol), PyBOP (34 mg, 0.0655 mmol), and Et N (34 μL,
.245 mmol) in DMF (500 μL) was stirred at 0 °C (1.5 h). The
(
3
39
(
(methylsulfonyl)oxy)hexanoate (323 mg, 1.36 mmol) was added to
a suspension of D-amphetamine·0.5H SO (200 mg, 1.09 mmol) and
reaction was quenched with H
(5 min) before the solvent was removed in vacuo. Purification by
preparative RP-HPLC (R = 39.0 min) followed by lyophilization gave
the title compound as a colorless oil (22 mg, 74%), [α]
CHCl ); δ (CD OD, 600 MHz, mixture of rotamers) 7.38−7.33 (m,
O (10 μL) and the reaction stirred at rt
2
2
4
K CO (450 mg, 3.26 mmol) in MeCN (7 mL), and the resulting
2
3
mixture was heated to 80 °C (15 h). Upon cooling, the solvent was
removed in vacuo, MeOH (3 mL) and 4 M aqueous LiOH (1 mL,
.00 mmol) were added, and the solution stirred at rt (5 h).
Purification by preparative RP-HPLC (R = 34.4 min) followed by
t
2
5
+ 3.3 (c 2.0,
D
4
3
H
3
2H), 7.30−7.25 (m, 3H), 3.83 (s, 2H), 3.72 (s, 2H), 3.53−3.45 (m,
1H), 3.36−3.34 (m, 0.5H), 3.27−3.22 (m, 2H), 3.22−3.19 (m, 0.5H),
3.11−3.01 (m, 2H), 2.73−2.67 (m, 1H), 1.75−1.67 (m, 2H), 1.62−
t
lyophilization gave the title compound as a colorless oil (352 mg,
5
8
7
3
1
(
1
9%), [α]2 + 5.8 (c 1.0, MeOH); δ (CD OD, 600 MHz) 7.35 (t, J =
D
H
3
.5 Hz, 2H), 7.30−7.25 (m, 3H), 4.89 (s, 2H), 3.52−3.45 (m, 1H),
.21 (dd, J = 13.3, 4.5 Hz, 1H), 3.12−3.02 (m, 2H), 2.70 (dd, J = 13.3,
0.1 Hz, 1H), 2.34 (t, J = 7.3 Hz, 2H), 1.76−1.69 (m, 1H), 1.69−1.63
1.55 (m, 2H), 1.48−1.39 (m, 11H), 1.24−1.20 (m, 3H); δ (CD OD,
C 3
151 MHz, mixture of rotamers) 173.4, 171.7, 162.3 (q, J = 35.3 Hz),
158.9, 137.3, 130.4, 130.0, 128.4, 117.9 (q, J = 292.3 Hz), 81.0, 56.9,
46.2, 45.1, 43.6, 40.3, 39.8, 29.6, 28.7, 27.0, 24.6, 16.0. Found:
435.2966. C H N O requires 435.2966.
m, 2H), 1.48−1.42 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H); δ (CD OD,
C
3
51 MHz) 177.2, 162.9 (q, J = 35.3 Hz), 137.3, 130.4, 129.9, 128.3,
23
39
4
4
E
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Journal of Medicinal Chemistry
Article
(
S)-2-Amino-N-(2-oxo-2-((5-((1-phenylpropan-2-yl)amino)-
(S)-(6-((1-Phenylpropan-2-yl)amino)hexanoyl)glycylglycine,
pentyl)amino)ethyl)acetamide, 8. A solution of carbamate 7 (22
10. Et N (41 μL, 0.295 mmol) was added to a solution of 2 (20 mg,
3
mg, 0.0413 mmol) in 4 M HCl in 4 M HCl in dioxane/MeOH (5:3,
0.0491 mmol), and methyl((aminoacetyl)amino)acetate·HCl (27 mg,
0.147 mmol) in DMF (300 μL) and stirred at rt (5 min) before the
addition of PyBOP (38 mg, 0.0736 mmol) and the solution stirred at
8
00 μL) was stirred at rt (1 h) before the solvent was removed in
vacuo to give the title compound as a colorless oil (16.8 mg, 100%),
2
5
[
α] + 3.7 (c 1.5, CHCl ); δ (CDCl , 600 MHz) 7.37 (t, J = 7.3 Hz,
rt (1 h). The reaction was quenched with H O (20 μL) and the
2
D
3
H
3
2
3
1
H), 7.33−7.28 (m, 2H), 3.95 (s, 2H), 3.81 (s, 2H), 3.53 (s, 1H),
.32−3.25 (m, 3H), 3.10 (s, 2H), 2.76 (t, J = 11.0, 1H), 1.78 (s, 2H),
.62 (s, 2H), 1.47 (s, 2H), 1.25 (d, J = 5.1 Hz, 2H); δ (CDCl , 151
reaction stirred at rt (10 min) before the solvent was removed in
vacuo. The residue was dissolved in MeOH (500 μL), 4 M aqueous
LiOH (200 μL, 0.800 mmol) added, and the reaction stirred at rt (2.5
C
3
MHz, mixture of rotamers) 171.2, 167.9, 137.4, 130.4, 129.9, 128.3,
3.5, 72.4, 62.1, 57.0, 46.3, 43.4, 41.7, 40.2, 40.0, 29.7, 27.1, 24.8, 16.0.
Found: 335.2442. C H N O requires 335.2441.
h). Purification by preparative RP-HPLC (R
t
= 31.7 min) followed by
lyophilization gave the title compound as a white solid (6 mg, 25%),
7
2
5
[
α] + 4.5 (c 0.6, MeOH); δ (CD OD, 600 MHz) 7.35 (t, J = 7.5
H 3
D
18
31
4
2
Hz, 2H), 7.30−7.25 (m, 3H), 3.93 (s, 2H), 3.92 (s, 2H), 3.52−3.46
(m, 1H), 3.21 (dd, J = 13.3, 4.4 Hz, 1H), 3.13−3.04 (m, 2H), 2.69
(dd, J = 13.2, 10.1 Hz, 1H), 2.32 (t, J = 7.2 Hz, 2H), 1.76−1.67 (m,
(
S)-2-Methyl-10,13,16-trioxo-1-phenyl-3,9,12,15-tetraazai-
cosan-20-oic Acid, 4. PyBOP (29 mg, 0.0552 mmol) and Et N (23
3
μL, 0.166 mmol) were added to a solution of monomethylglutarate (7
μL, 0.0552 mmol) in DMF (200 μL) and stirred at rt (5 min) before
the addition of a solution of amine 8 (15 mg, 0.0368 mmol) in DMF
4H), 1.47 (p, J = 7.7 Hz, 2H), 1.22 (d, J = 6.6 Hz, 3H); δ (CD OD,
C 3
1
1
2
3
51 MHz) 176.3, 172.8, 172.1, 162.6 (q, J = 36.2 Hz), 137.3, 130.4,
30.0, 128.4, 118.1 (q, J = 290.6 Hz), 57.0, 46.1, 43.2, 41.8, 40.3, 36.2,
7.1, 26.9, 25.7, 16.0. Found: 364.2231. C H N O requires
(
100 μL) and the solution stirred at rt (45 min). The reaction was
quenched with H O (50 μL) and the reaction stirred at rt (10 min)
19 30
3
4
2
64.2231.
S)-3-((1-Phenylpropan-2-yl)amino)propanoic Acid, 11. tert-
Butyl 3-bromo-propionate (25 mg, 0.119 mmol) was added to a
suspension of D-amphetamine·0.5H SO (20 mg, 0.109 mmol) and
before the solvent was removed in vacuo. The residue was dissolved in
MeOH (500 μL), 4 M aqueous LiOH (100 μL) added, and the
reaction stirred at rt (1.5 h). Purification by preparative RP-HPLC (Rt
(
=
33.4 min) followed by lyophilization gave the title compound as a
2
4
2
5
K CO (45 mg, 0.329 mmol) in MeCN (700 μL), and the resulting
white solid (7.5 mg, 36%), [α] + 4.0 (c 0.75, CD OD); δ (CD OD,
6
2 3
D
3
H
3
mixture was heated to 80 °C (18 h). Upon cooling, the solvent was
removed in vacuo and purification by preparative TLC using MeOH/
CH Cl (1:9) gave the intermediate ester (16 mg, 56%). A solution of
00 MHz) 7.36 (t, J = 7.6 Hz, 2H), 7.30−7.25 (m, 3H), 3.84−3.82 (m,
4
H), 3.53−3.47 (m, 1H), 3.25 (t, J = 6.8, 2H), 3.21 (dd, J = 13.4, 4.6
Hz, 1H), 3.11−3.02 (m, 2H), 2.70 (dd, J = 13.2, 10.0 Hz, 1H), 2.36 (q,
J = 7.6 Hz, 4H), 1.94−1.89 (m, 2H), 1.74−1.67 (m, 2H), 1.59 (p, J =
2
2
intermediate ester (16 mg, 0.0607 mmol) in TFA/CH Cl (500 μL)
2
2
was stirred (2 h), whereupon the solvent was removed in vacuo to give
6
.9 Hz, 2H), 1.43 (p, J = 7.7 Hz, 2H), 1.22 (d, J = 6.5 Hz, 3H); δC
25
the title compound as a colorless gum (19 mg, 97%), [α]D + 13.6 (c
(
CD OD, 151 MHz, mixture of rotamers) 176.6, 176.5, 172.6, 171.7,
3
1
7
3
2
.5, CHCl ); δ (CHCl , 600 MHz) 9.92 (br s, 2H), 8.44 (br s, 2H),
3 H 3
1
61.9 (q, J = 38.6, 36.9 Hz), 137.3, 130.4, 130.0, 128.4, 128.4, 117.7
.30 (t, J = 7.3 Hz, 2H), 7.26−7.23 (m, 1H), 7.18 (d, J = 7.3 Hz, 2H),
.52 (br s, 1H), 3.36 (br s, 1H), 3.30−3.22 (m, 2H), 2.92−2.87 (m,
H), 2.83−2.77 (m, 1H), 1.32 (d, J = 6.4 Hz, 3H); δ (CHCl , 151
(
d, J = 293.0 Hz), 56.9, 46.2, 44.2, 43.7, 40.3, 39.8, 35.7, 34.1, 29.6,
2
4
7.0, 24.6, 21.9, 16.0. Found: 449.2758. C H N O requires
23 37 4 5
C
3
49.2758.
S)-4-Oxo-4-((1-phenylpropan-2-yl)amino)butanoic Acid, 5.
MHz) 174.5, 161.7 (q, J = 38.2 Hz), 135.4, 129.3, 129.1, 127.6, 115.9
(q, J = 291.6, 290.8 Hz), 57.1, 41.2, 39.3, 30.3, 15.7. Found: 208.1332.
C H NO requires 208.1332.
(
25
According to the method of Kosten, a solution of (+)-methamphet-
amine·HCl (30 mg, 0.162 mmol), succinic anhydride (24 mg, 0.242
mmol), and Et N (68 μL, 0.485 mmol) in CH Cl (1 mL) was heated
to 50 °C (1 h) before the solvent was removed in vacuo. Purification
by preparative RP-HPLC (R = 40.0 min) followed by lyophilization
12
18
2
(
S)-(3-((1-Phenylpropan-2-yl)amino)propanoyl)glycine, 12.
3
2
2
EDC·HCl (4 mg, 0.0205 mmol) and Et N (6 μL, 0.0411 mmol)
3
were added to a solution of 11 (4.4 mg, 0.0137 mmol) in CH Cl (400
2
2
t
μL) and stirred at rt (5 min) before the addition of a solution of tert-
butyl glycinate (67 mg, 0.242 mmol) in CH Cl (200 μL) and the
25
gave the title compound as a colorless oil (35 mg, 87%), [α]D + 34.9
2
2
(c 2.0, CDCl ); δH (CD OD, 600 MHz, mixture of rotamers) 7.30−
3
3
solution stirred at rt (1 h). The reaction was quenched with H O (10
2
7.17 (m, 3H), 7.15 (d, J = 7.2 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 5.01−
4.93 (m, 0.5H), 4.13−4.06 (m, 0.5H), 2.86 (s, 1.5H), 2.82−2.76 (m,
2H), 2.76−2.69 (m, 1.5H), 2.61−2.53 (m, 1.5H), 2.50−2.42 (m,
1.5H), 2.38−2.31 (m, 0.5H), 2.07−2.00 (m, 0.5H), 1.26 (d, J = 6.7 Hz,
1.5H), 1.11 (d, J = 6.8 Hz, 1.5H); δ (CD OD, 151 MHz, mixture of
μL) and the reaction stirred at rt (5 min) before the solvent was
removed in vacuo. Purification by preparative TLC using MeOH/
CH Cl (1:9) gave the intermediate ester. A solution of intermediate
2
2
ester in TFA/CH Cl (500 μL) was stirred (2 h), whereupon the
2
2
C
3
solvent was removed in vacuo to give the title compound as a colorless
rotamers) 177.1, 177.0, 172.3, 172.2, 138.2, 138.0, 128.9, 128.9, 128.8,
1
2
25
gum (2.1 mg, 90%), [α]D + 5.5 (c 0.2, CH OH); δH (CD OD, 600
3
3
28.4, 127.0, 126.5, 54.8, 50.3, 40.6, 40.1, 29.7, 29.7, 29.4, 28.8, 27.8,
7.0, 18.8, 17.2. Found: 250.1437. C H NO requires 250.1438.
MHz) 7.39−7.36 (m, 2H), 7.32−7.28 (m, 3H), 3.97 (s, 2H), 3.60−
14
20
3
3.54 (m, 1H), 3.45−3.34 (m, 2H), 3.21 (dd, J = 13.4, 5.0 Hz, 1H),
(
S)-(6-((1-Phenylpropan-2-yl)amino)hexanoyl)glycine, 9.
2
.81−2.74 (m, 3H), 1.27 (d, J = 6.5 Hz, 3H); δ (CD OD, 151 MHz)
C
3
Et N (41 μL, 0.295 mmol) was added to a solution of 2 (20 mg,
3
173.2, 172.5, 137.1, 130.4, 130.0, 128.4, 57.1, 42.3, 41.7, 40.3, 31.7,
0
.0491 mmol), and glycine methyl ester·HCl (18 mg, 0.147 mmol) in
DMF (300 μL) and stirred at rt (5 min) before the addition of PyBOP
38 mg, 0.0736 mmol) and the solution stirred at rt (1 h). The
reaction was quenched with H O (20 μL) and the reaction stirred at rt
1
6.0. Found: 265.1547. C H N O requires 265.1547.
Conjugation of Haptens. Acids. A solution of hapten (150 mM),
14
21
2
3
(
EDC·HCl (4.5 equiv), and sulfo-NHS (1.5 equiv) in 10% H O/DMF
2
2
was agitated at rt until complete activation was observed by LCMS
analysis (adding further EDC·HCl if necessary). DT or BSA in PBS
(
10 min) before the solvent was removed in vacuo. The residue was
dissolved in MeOH (500 μL), 4 M aqueous LiOH (200 μL, 0.800
mmol) added, and the reaction stirred at rt (2.5 h). Purification by
preparative RP-HPLC (R = 32.8 min) followed by lyophilization gave
the title compound as a white solid (14.5 mg, 70%), [α] + 4.4 (c 0.5,
MeOH); δ (CD OD, 600 MHz) 7.38 (t, J = 7.4 Hz, 2H), 7.33−7.27
(
pH 7.4) was added to the activated hapten 2−5, 9, 10, or 12 (ca. 250
equiv for DT, 1500 equiv for TT) to give a final concentration of 3
mg/mL in PBS (pH 7.4) and the solutions mixed (rt, 6−8 h, then 4
°C, 16 h).
t
2
5
D
H
3
Thiol. A solution of sulfo-GMBS (0.5 mg per mg protein) and BSA
(5 mg/mL in PBS (pH 7.4)) were mixed (rt, 1.5 h) before excess
sulfo-GMBS was removed (Zeba desalting column). A solution of
(
1
2
3
m, 3H), 3.93 (d, J = 1.3 Hz, 2H), 3.54−5.48 (m, 1H), 3.23 (dd, J =
3.4, 4.5 Hz, 1H), 3.15−3.05 (m, 2H), 2.72 (m, 1H), 2.35−2.31 (m,
H), 1.78−1.68 (m, 4H), 1.49 (p, J = 7.7 Hz, 2H), 1.24 (d, J = 6.4 Hz,
H); δ (CD OD, 151 MHz) 174.4, 171.2, 159.7 (q, J = 37.0 Hz),
2
2
hapten 1 (ca. 250 equiv) in 10% DMSO in maleimide conjugation
buffer (Imject, Thermo Pierce) was added dropwise to the activated
BSA solution to give a final concentration of 2 mg/mL protein, which
was mixed (rt, 6 h, then 4 °C, 16 h). The resulting protein conjugates
were dialyzed into PBS (pH 7.4) at 4 °C. DT and TT conjugates were
C
3
1
3
35.4, 128.5, 128.1, 126.5, 115.6 (q, J = 289.2 Hz), 55.0, 44.2, 39.8,
8.4, 34.2, 25.2, 25.0, 24.0, 14.1. Found: 307.2017. C H N O
1
7
27
2
3
requires 307.2016.
F
DOI: 10.1021/acs.jmedchem.6b00084
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
used for immunization; BSA conjugates were used for ELISA plate
coating.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00084.
■
*
S
Active Immunization Protocol. All procedures adhered to the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals and were approved by the Institutional Animal
Care and Use Committee of The Scripps Research Institute. No
adverse reactions to the vaccines were observed and all mice
maintained a healthy weight throughout the vaccine trial. 2-DT, 3-
DT, 4-DT, 5-DT, 9-DT, 10-DT, 12-DT, and 12-TT in PBS (pH 7.4)
Additional figures showing hapten densities, antibody
titers, binding constants and concentrations, NMR data
for haptens 1 and 3, NMR spectra, and MALDI traces.
(PDF)
(
100 μL, 1 mg/mL for DT conjugates; 75 μL, 1 mg/mL for TT
conjugates) formulated with Sigma Adjuvant System (SAS, 100 μL) or
Alhydrogel (Invivogen, 10 mg/mL) (100 μL for DT conjugates, 75 μL
for TT conjugates) with or without phosphorothioated CpG ODN
AUTHOR INFORMATION
■
Corresponding Author
E-mail: kdjanda@scripps.edu. Phone: (858) 784-2516. Fax:
858) 784-2595.
1
826 (Eurofins MWG Operon) (75 μL) were used to immunize
*
groups of Swiss Webster mice (n = 6) via subcutaneous injection on
days 0, 21, and 35 for DT conjugates or days 0, 14, and 28 for TT
conjugates. Serum was collected via tail bleed on days 28 and 42 and
via cardiac bleed on day 63.
(
Notes
The authors declare no competing financial interest.
ELISA. Production of anti-MA IgG was evaluated by ELISA.
Microtiter plates (Costar 3690) were incubated with coating antigen
ACKNOWLEDGMENTS
■
We acknowledge the support of The Scripps Research Institute,
Skaggs Institute for Chemical Biology, and the National
Institute on Drug Abuse under grants R01-DA024705 and
F31-DA037709. This is manuscript #29277 from The Scripps
Research Institute.
1-BSA in PBS (5 μg/mL, 25 μL) (18 h, 37 °C). Then, 5% nonfat milk
in PBS (30 min, 37 °C) was added to block nonspecific binding.
Mouse sera in 1% BSA were serially diluted across the plate before
incubation in a moist chamber (1.5 h, 37 °C). The plate was washed
with dH O or PBS before incubation with peroxidase-conjugated
2
donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.)
in a moist chamber (30 min, 37 °C). The plates were further washed
ABBREVIATIONS USED
BCA, bicinchoninic acid assay; BSA, bovine serum albumin;
CpG ODN, cytosine-guanine oligodeoxynucleotide; dH O,
deionized water; DMF, dimethylformamide; DT, diphtheria
toxoid; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide; ELISA, enzyme-linked immunosorbent assay;
■
with dH O or PBS before being developed with the TMB substrate kit
2
(
Thermo Pierce) and the absorbance at 450 nm measured on a
2
e
microplate reader (SpectraMax M2 Molecular Devices). Titers were
calculated as the dilution corresponding to 50% of the maximum
absorbance from a plot of the absorbance versus log(dilution) using
GraphPad Prism 6.
Ig, immunoglobulin; K , dissociation constant; KLH, keyhole
d
Radioimmunoassay. Dissociation constants and antibody con-
centrations were determined using competitive RIA. Mouse sera were
pooled and diluted into 2% BSA, giving a concentration that was
determined to bind ca. 30−40% (+)-[2′,6′- H(n)] methamphetamine
tracer (20 000 dpm, 39 Ci/mmol (National Institute on Drug Abuse,
limpet hemocyanin LCMS, liquid chromatography mass
spectrometry; MA, methamphetamine; MALDI-MS, matrix-
assisted laser desorption/ionization mass spectrometry;
MWCO, molecular weight cutoff; NMR, nuclear magnetic
resonance; PBS, phosphate-buffered saline; PyBOP, benzo-
triazol-1-yl-oxytripyrrolidinophosphonium hexafluorophos-
phate; RIA, radioimmunoassay; RP-HPLC, reversed-phase
high-performance liquid chromatography; SAS, Sigma Adjuvant
System; SEM, standard error of the mean; sulfo-GMBS, N-(γ-
maleimidobutyryloxy) sulfosuccinimide ester; sulfo-NHS, N-
hydroxysulfosuccinimide; TFA, trifluoroacetic acid; TLC, thin-
layer chromatography; TMB, 3,3′,5,5′-tetramethylbenzidine;
TT, tetanus toxoid; UV, ultraviolet
3
Bethesda, MD)). Diluted serum (60 μL) and methamphetamine tracer
(
60 μL) were added to the sample chamber of a 5 kDa MWCO 6-well
Equilibrium Dialyzer (Harvard Apparatus), and unlabeled (+)-meth-
amphetamine (120 μL) at varying concentrations in 1% BSA was
added to the buffer chamber. After equilibration on a plate rotator
(
(
Harvard Apparatus) at rt (22−26 h), a sample from each chamber
60 μL) was diluted into Ecolite(+) liquid scintillation cocktail (5 mL,
MP Biomedicals) and the radioactivity measured using a Beckman LS
500 Scintillation Counter. K ’s and antibody concentrations were
6
d
4
0
calculated according to Mu
̈
ller.
REFERENCES
■
Locomotion. Mice were allowed to acclimate for 1 h in a plastic
cage (10.5 in. × 19 in. × 8 in.) with clear ventilated acrylic top. Mice
were then quickly removed and injected with saline or 0.5, 1, 2, or 4
mg/kg MA before being returned to their cages; cages were wiped
down with dry paper towels to remove excess debris while mice were
being injected. The mice were then returned to the cage to be
recorded and tracked by overhead camera using ANY-Maze video
tracking software (Stoelting Co., Wood Dale, IL). Sessions were run
(
1) Sulzer, D.; Sonders, M. S.; Poulsen, N. W.; Galli, A. Mechanisms
of Neurotransmitter Release by Amphetamines: A Review. Prog.
Neurobiol. 2005, 75, 406−433.
(2) Rawson, R. A. Treatment for Stimulant Use Disorders; Center for
Substance Abuse Treatment, Substance Abuse and Mental Health
Services Administration: Rockville, MD, 1999.
(3) Rawson, R. A.; Anglin, M. D.; Ling, W. Will the Methamphet-
amine Problem Go Away? J. Addict. Dis. 2001, 21, 5−19.
(4) Vocci, F. J.; Appel, N. M. Approaches to the Development of
Medications for the Treatment of Methamphetamine Dependence.
Addiction 2007, 102, 96−106.
2
during the middle of the dark cycle in a 4.6 × 4.6 m room with a
single 30 W upward-directed light source, and repeated on mice after a
two-day washout period until all mice received all MA doses. Distance
traveled (m) and time spent immobile (s; 80% pixel consistency for at
least 5 s threshold) were measured and analyzed via Prism 6 software,
using a two-way repeated-measures ANOVA with Bonferroni posthoc
corrected comparisons. Significance was set at α = 0.05 and data is
represented as mean ± SEM.
(5) Chen, Y.-H.; Chen, C.-H. The Development of Antibody-based
Immunotherapy for Methamphetamine Abuse: Immunization, and
Virus-Mediated Gene Transfer Approaches. Curr. Gene Ther. 2013, 13,
3
9−50.
(6) Carroll, K. M.; Onken, L. S. Behavioral Therapies for Drug
Abuse. Am. J. Psychiatry 2005, 162, 1452−1460.
G
DOI: 10.1021/acs.jmedchem.6b00084
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
7) McKetin, R.; Najman, J. M.; Baker, A. L.; Lubman, D. I.; Dawe,
(24) Ru
̈
edi-Bettschen, D.; Wood, S. L.; Gunnell, M. G.; West, C. M.;
S.; Ali, R.; Lee, N. K.; Mattick, R. P.; Mamun, A. Evaluating the Impact
of Community-based Treatment Options on Methamphetamine Use:
Findings from the Methamphetamine Treatment Evaluation Study
Pidaparthi, R. R.; Carroll, F. I.; Blough, B. E.; Owens, S. M.
Vaccination Protects Rats from Methamphetamine-induced Impair-
ment of Behavioral Responding for Food. Vaccine 2013, 31, 4596−
4602.
(
MATES). Addiction 2012, 107, 1998−2008.
8) Gonzales, R.; Mooney, L.; Rawson, R. A. The Methamphetamine
Problem in the United States. Annu. Rev. Public Health 2010, 31, 385−
98.
9) Nicosia, N.; Pacula, R. L.; Kilmer, B.; Lundberg, R.; Chiesa, J. The
(
(25) Shen, X. Y.; Kosten, T. A.; Lopez, A. Y.; Kinsey, B. M.; Kosten,
T. R.; Orson, F. M. A Vaccine against Methamphetamine Attenuates
its Behavioral Effects in Mice. Drug Alcohol Depend. 2013, 129, 41−48.
(26) Collins, K. C.; Schlosburg, J. E.; Lockner, J. W.; Bremer, P. T.;
Ellis, B. A.; Janda, K. D. Lipid Tucaresol as an Adjuvant for
Methamphetamine Vaccine Development. Chem. Commun. 2014, 50,
4079−4081.
(27) Collins, K. C.; Janda, K. D. Investigating Hapten Clustering as a
Strategy to Enhance Vaccines against Drugs of Abuse. Bioconjugate
Chem. 2014, 25, 593−600.
3
(
Economic Cost of Methamphetamine Use in the United States, 2005;
RAND Corporation: Santa Monica, CA, 2009. http://www.rand.org/
pubs/monographs/MG829 (accessed September 27, 2015).
(
10) Janeway, C. A.; Travers, P.; Walport, M.; Shlomchik, M. J.
Chapter 9. The Humoral Immune Response; B-cell Activation by
Armed Helper T Cells. In Immunobiology: The Immune System in
Health and Disease; Garland Science: New York, 2001.
(28) Pravetoni, M.; Le Naour, M.; Harmon, T. M.; Tucker, A. M.;
Portoghese, P. S.; Pentel, P. R. An Oxycodone Conjugate Vaccine
Elicits Drug-specific Antibodies that Reduce Oxycodone Distribution
to Brain and Hot-plate Analgesia. J. Pharmacol. Exp. Ther. 2012, 341,
(
11) Cornuz, J.; Zwahlen, S.; Jungi, W. F.; Osterwalder, J.; Klingler,
K.; van Melle, G.; Bangala, Y.; Guessous, I.; Muller, P.; Willers, J.;
Maurer, P.; Bachmann, M. F.; Cerny, T. A Vaccine Against Nicotine
for Smoking Cessation: A Randomized Controlled Trial. PLoS One
̈
2
(
25−232.
29) Pravetoni, M.; Le Naour, M.; Tucker, A. M.; Harmon, T. M.;
2
(
008, 3, e2547.
12) Hatsukami, D. K.; Jorenby, D. E.; Gonzales, D.; Rigotti, N. A.;
Hawley, T. M.; Portoghese, P. S.; Pentel, P. R. Reduced
Antinociception of Opioids in Rats and Mice by Vaccination with
Immunogens Containing Oxycodone and Hydrocodone Haptens. J.
Med. Chem. 2013, 56, 915−923.
Glover, E. D.; Oncken, C. A.; Tashkin, D. P.; Reus, V. I.; Akhavain, R.
C.; Fahim, R. E. F.; Kessler, P. D.; Niknian, M.; Kalnik, M. W.;
Rennard, S. I. Immunogenicity and Smoking-Cessation Outcomes for
a Novel Nicotine Immunotherapeutic. Clin. Pharmacol. Ther. 2011, 89,
(30) Arora, R.; Kosten, T. A.; Bennett, R. S.; Kinsey, B. M.; Orson, F.
M.; Kosten, T. R. Preclinical Efficacy of an Anti-methamphetamine
Vaccine with an E6020 Adjuvant. Drug Alcohol Depend. 2015, 146,
e211.
3
(
92−399.
13) Martell, B. A.; Orson, F. M.; Poling, J.; Mitchell, E.; Rossen, R.
D.; Gardner, T.; Kosten, T. R. Cocaine Vaccine for the Treatment of
Cocaine Dependence in Methadone-maintained Patients: A Random-
ized, Double-blind, Placebo-controlled Efficacy Trial. Arch. Gen.
Psychiatry 2009, 66, 1116−1123.
(31) Leit De Moradei, S. M.; Tessier, P.; Smil, D.; Wahhab, A.;
Deziel, R.; Manku, S.; Mancuso, J.; Therrien, E.; Allan, M.; Chantihny,
Y. A.; Ajamian, A.; Beaulieu, P. Inhibitors of Histone Deacetylase. WO
1
(
02760 (A1), 2006.
32) Cai, X.; Tsuchikama, K.; Janda, K. D. Modulating Cocaine
Vaccine Potency through Hapten Fluorination. J. Am. Chem. Soc. 2013,
35, 2971−2974.
33) Cai, X.; Whitfield, T.; Hixon, M. S.; Grant, Y.; Koob, G. F.;
(
14) Kosten, T. R.; Biegel, D. Therapeutic Vaccines for Substance
Dependence. Expert Rev. Vaccines 2002, 1, 365−371.
15) Martell, B. A.; Mitchell, E.; Poling, J.; Gonsai, K.; Kosten, T. R.
(
1
(
Vaccine Pharmacotherapy for the Treatment of Cocaine Dependence.
Biol. Psychiatry 2005, 58, 158−164.
(
R. W. Cocaine-Specific Antibodies Blunt the Subjective Effects of
Smoked Cocaine in Humans. Biol. Psychiatry 2010, 67, 59−65.
(
Janda, K. D. Probing Active Cocaine Vaccination Performance through
Catalytic and Noncatalytic Hapten Design. J. Med. Chem. 2013, 56,
16) Haney, M.; Gunderson, E. W.; Jiang, H.; Collins, E. D.; Foltin,
3
(
701−3709.
34) Lockner, J. W.; Ho, S. O.; McCague, K. C.; Chiang, S. M.; Do,
17) Hatsukami, D. K.; Rennard, S.; Jorenby, D.; Fiore, M.;
T. Q.; Fujii, G.; Janda, K. D. Enhancing Nicotine Vaccine
Immunogenicity with Liposomes. Bioorg. Med. Chem. Lett. 2013, 23,
Koopmeiners, J.; de Vos, A.; Horwith, G.; Pentel, P. R. Safety and
Immunogenicity of a Nicotine Conjugate Vaccine in Current Smokers.
Clin. Pharmacol. Ther. 2005, 78, 456−467.
9
(
75−978.
35) Bremer, P. T.; Schlosburg, J. E.; Lively, J. M.; Janda, K. D.
(
18) Kinsey, B. M.; Jackson, D. C.; Orson, F. M. Anti-drug Vaccines
to Treat Substance Abuse. Immunol. Cell Biol. 2009, 87, 309−314.
19) Byrnes-Blake, K. A.; Carroll, F. I.; Abraham, P.; Owens, S. M.
Injection Route and TLR9 Agonist Addition Significantly Impact
Heroin Vaccine Efficacy. Mol. Pharmaceutics 2014, 11, 1075−1080.
(
(36) Still, W. C.; Kahn, M.; Mitra, A. Rapid Chromatographic
Generation of Anti-(+)methamphetamine Antibodies is Not Impeded
by (+)Methamphetamine Administration During Active Immunization
of Rats. Int. Immunopharmacol. 2001, 1, 329−338.
Technique for Preparative Separations with Moderate Resolution. J.
Org. Chem. 1978, 43, 2923−2925.
(37) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.;
(
20) Duryee, M. J.; Bevins, R. A.; Reichel, C. M.; Murray, J. E.; Dong,
Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.;
Olson, B. J.; Klenk, D. C. Measurement of Protein using Bicinchoninic
Acid. Anal. Biochem. 1985, 150, 76−85.
Y.; Thiele, G. M.; Sanderson, S. D. Immune Responses to
Methamphetamine by Active Immunization with Peptide-based,
Molecular Adjuvant-containing Vaccines. Vaccine 2009, 27, 2981−
(38) Wengatz, I.; Schmid, R. D.; Kreißig, S.; Wittmann, C.; Hock, B.;
2
(
988.
21) Carroll, F. I.; Blough, B. E.; Pidaparthi, R. R.; Abraham, P.;
Ingendoh, A.; Hillenkamp, F. Determination of the Hapten Density of
Immuno-conjugates by Matrix-assisted UV Laser Desorption/Ioniza-
tion Mass Spectrometry. Anal. Lett. 1992, 25, 1983−1997.
Gong, P. K.; Deng, L.; Huang, X.; Gunnell, M.; Lay, J. O.; Peterson, E.
C.; Owens, S. M. Synthesis of Mercapto-(+)-methamphetamine
Haptens and Their Use for Obtaining Improved Epitope Density on
(39) Ekwuribe, N. N.; Price, C. H.; Ansari, A. M.; Radhakrishnan, B.;
Odenbaugh, A. L. Mixtures of Insulin Drug-oligomer Conjugates
Comprising Polyalkylene Glycol, Uses thereof, and Methods of
Making Same. WO 0027748 (A1), 2003.
(
+)-Methamphetamine Conjugate Vaccines. J. Med. Chem. 2011, 54,
221−5228.
22) Moreno, A. Y.; Mayorov, A. V.; Janda, K. D. Impact of Distinct
5
(
(
̈
40) Muller, R. Determination of Affinity and Specificity of Anti-
Chemical Structures for the Development of a Methamphetamine
Vaccine. J. Am. Chem. Soc. 2011, 133, 6587−6595.
(
hapten Antibodies by Competitive Radioimmunoassay. Methods
Enzymol. 1983, 92, 589−601.
23) Miller, M. L.; Moreno, A. Y.; Aarde, S. M.; Creehan, K. M.;
Vandewater, S. A.; Vaillancourt, B. D.; Wright, M. J., Jr; Janda, K. D.;
Taffe, M. A. A Methamphetamine Vaccine Attenuates Methamphet-
amine-Induced Disruptions in Thermoregulation and Activity in Rats.
Biol. Psychiatry 2013, 73, 721−728.
H
DOI: 10.1021/acs.jmedchem.6b00084
J. Med. Chem. XXXX, XXX, XXX−XXX